Bumble bees are important alternative pollinators and model insects due to their highly developed sociality and colony management. In order to better understand their molecular mechanisms, studies focusing on the genetic and molecular aspects of their development and behavior are needed. Although quantitative real-time polymerase chain reaction (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different lines and tissues) must first be identified to ensure the accurate normalization of target genes. In order to contribute to molecular studies on bumble bees, we used Bombus terrestris to determine the expression stability of eight reference genes (β-actin (ACT), Arginine Kinase (AK), Phospholipase A2 (PLA2), Elongation factor 1 alpha (EF-1), Ribosomal proteins (S5, S18, S28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in five different lines and several tissues (ovary, thorax, fat body, and head) using RT-qPCR procedures and four analysis programs (RefFinder, NormFinder, BestKeeper, and geNorm). In general, the S28, S5, and S18 ribosomal protein genes and the PLA2 and EF-1 genes showed the highest stability and were therefore identified as suitable reference genes for the bumble bee species and their defined lines and tissues. Our results also emphasized the need to evaluate the stability of candidate reference genes for any differently designed lines and tissue conditions in bumble bee species.

Bumblebees (B. terrestris) play a crucial role as highly efficient biological agents in commercial pollination. Understanding the molecular mechanisms governing their adaptation to diverse seasonal environments may pave the way for effective management strategies in the future. With the burgeoning advancement in post-genetic studies focusing on B. terrestris, there is a critical need to normalize quantitative real-time PCR (qRT-PCR) data using suitable reference genes. To address this necessity, we employed RefFinder, a software-based tool, to assess the suitability of several candidate endogenous control genes, including actin (ACT), arginine kinase (AK), elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate (GAPDH), phospholipase (PLA2), and ribosomal proteins (S18, S28). These genes were evaluated for their efficacy as biological endogenous controls by examining their expression patterns across various environmental conditions corresponding to different seasons (Spring, Summer, Autumn, Winter) and tissues (ovary, fat body, thorax, head) in bumblebees. Moreover, the study investigated the significance of selecting appropriate reference genes for three key genes involved in the juvenile hormone (JH) signaling pathways: Krüppel homolog 1 (Kr-h1), methyl farnesoate epoxidase (MFE), and Vitellogenin (Vg). Our research identifies specific genes suitable for normalization in B. terrestris, thereby offering valuable insights into gene expression and functional metabolic genetics under varying seasonal conditions. This catalog of reference genes will serve as a valuable resource for future research endeavors.